The 1956 paper A simple ultraviolet spectrophotometric method for the determination of protein is constantly referenced as the one who provided a method for peptide concentration calculation that does not need aromatic residues in the primary structure. The formulas are:
A = (abs215 - abs225)*0.144
B = (abs205)*0.31
Concentration(mg/mL) = [(A+B)/2]*dilution_factor
However, I don't find the 1956 paper with the formula explanation available, and, as it looks like, neither other researchers, since they usually quote the Murphy and Kies 1960 paper that quotes Waddell's method.
Does anyone has a copy of 1956 Waddell paper? If not, could at least someone explain me the formula, item by item?
Thanks in advance.
Dear Bruno Santos ,
The confusion is caused by the fact that you refer to two different methods. One formula originates from the Scopes method:
Protein concentration (mg/ml) = (Absorbance at 205 nm)/31 where 31 is the rough measure for the extinction coefficient (https://assets.thermofisher.com/TFS-Assets/MSD/Application-Notes/AN52774-quantify-protein-peptide-preparations-205-nm.pdf )
The other formula is from the Waddel method:
Protein concentration (μg/mL) = 144 (A215 – A225) or
Protein concentration (mg/mL) = 0.144 (A215 – A225)
I presume that the third formula is ‘simply’ taken the average value of the two different methods.
Hopes this clarifies the matter somewhat.
Best regards.
PS. I see numerous comments that the Waddel method is limited to experiments using buffers with little to no absorbance in the 205-225 nm region and that Abs215 < 1.5 (see for example enclosed file).
Thank you, Rob Keller .
I took a look in the Thermo Fisher document you have appended, and, if I understood correctly, the Scope method is actually an improvement of the a205*31. Instead of using 31 as a general extinction coefficient, it uses the ratio abs280/abs205. The original a205*31 comes from 1951 THE ULTRAVIOLET ABSORPTION SPECTRA OF PROTEINS
BY A. ROBERT GOLDFARB, LEO J. SAIDEL, AND ERWIN MOSOVICH (Article Ultraviolet Absorption Spectra of Proteins
). They have studied a few proteins and saw that their coefficients ranged from 30-34.This clarifies part of the formula. Thank you very much!
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